WO2023036043A1

WO2023036043A1 - Anti-cancer binding molecule and use thereof

Info

Publication number: WO2023036043A1
Application number: PCT/CN2022/116450
Authority: WO
Inventors: 孟祥雪; 史继亚; 介淼星; 杨敏; 王雅秋; 任志衡; 陈立模; 李文佳
Original assignee: 广东东阳光药业有限公司
Priority date: 2021-09-09
Filing date: 2022-09-01
Publication date: 2023-03-16
Also published as: CN115960242B; CN115960242A

Abstract

Provided are an anti-cancer binding molecule and the use thereof, and specifically provided is a bispecific binding molecule of 4-1BB and GPC3. The bispecific binding molecule comprises a first domain that specifically binds to 4-1BB, and a second domain that specifically binds to GPC3. Further provided is the use of the bispecific binding molecule in the treatment or prevention of cancers.

Description

Anticancer binding molecules and their applications

technical field

The disclosure belongs to the field of biotechnology, and in particular relates to a binding molecule specifically binding to 4-1BB and glypican 3 (GPC3) and an application thereof.

Background technique

4-1BB (also known as CD137, TNFRSF9, etc.) is a member of the tumor necrosis factor receptor superfamily (TNFRS). Antibodies against 4-1BB have the ability to activate the 4-1BB signaling pathway, and have potential medical value for tumor treatment, anti-infection, and anti-autoimmune diseases. Glypican 3 (GPC3) belongs to the carcinoembryonic antigen of the glypican family and is highly expressed in a variety of cancer cells, especially hepatocellular carcinoma (HCC), melanoma, and Wilms tumor and hepatoblastoma. Therefore, there is a clinical need for novel protein drugs based on 4-1BB activating antibodies that can target tumor cells and activate the 4-1BB pathway to treat tumors.

Contents of the invention

In order to solve one of the above-mentioned technical problems in the prior art, the present disclosure provides a new method for targeting the tumor-associated antigen GPC3 (glypican 3) and simultaneously activating the immune pathways related to the 4-1BB signaling pathway, which can Effectively solve the side effects of 4-1BB activating antibody.

In one aspect of the present disclosure, a binding molecule is provided, the binding molecule comprising: a first domain specifically binding to 4-1BB or a fragment thereof, and a first domain specifically binding to Glypican 3 (GPC3) or the second domain of a fragment thereof.

According to some embodiments of the present disclosure, the binding molecule further comprises a third domain comprising an Fc fragment of an immunoglobulin.

According to some embodiments of the present disclosure, the immunoglobulin is selected from IgA, IgG, IgM, IgD and IgE. In a specific embodiment of the present disclosure, the immunoglobulin is IgG, such as IgG1, IgG2, IgG3 or IgG4.

According to some embodiments of the present disclosure, the Fc fragment has one or more mutations among L234F, L235E, P331S, D356E and L358M, wherein the Fc fragment is numbered according to the EU index of Kabat.

In a specific embodiment of the present disclosure, the third domain has an amino acid sequence as shown in SEQ ID NO: 55, 62 or 69 or a variant thereof.

In some embodiments of the present disclosure, the binding molecule may include a heavy chain constant region CH ₁ linked to the N-terminus of the Fc fragment.

According to some embodiments of the present disclosure, the first domain comprises a first heavy chain variable region VH ₁ and a first light chain variable region VL ₁ . According to some embodiments of the present disclosure, the structure of the first domain is selected from Fab, Fab', F(ab') ₂ , Fv or scFv. For the first domain with Fv or scFv structure, the C-terminal of the VH ₁ peptide chain is directly connected to the N-terminal of the VL ₁ peptide chain or connected via a linker, and vice versa.

In some specific embodiments of the present disclosure, the VH ₁ comprises: (a) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively; or, (b) comprising HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO:7, 8 and 9. In some specific embodiments of the present disclosure, the _VL1 comprises: (c) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively; or, (d) comprising LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.

According to some specific embodiments of the present disclosure, the first structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 1, HCDR2 including the amino acid sequence shown in SEQ ID NO: 2, including the amino acid sequence shown in SEQ ID NO: 2 The HCDR3 of the amino acid sequence shown in ID NO:3, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:4, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence of LCDR3 is shown.

According to some specific embodiments of the present disclosure, the first structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 7, HCDR2 including the amino acid sequence shown in SEQ ID NO: 8, including the amino acid sequence shown in SEQ ID NO: 8 The HCDR3 of the amino acid sequence shown in ID NO:9, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:10, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:11, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:12 The amino acid sequence of LCDR3 is shown.

According to some embodiments of the present disclosure, the first domain of the binding molecule of the present disclosure comprises VH ₁ and VL ₁ . The VH ₁ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO:31 or 33. The VL ₁ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:32 or 34.

In a specific embodiment of the present disclosure, the VH ₁ of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 31, and VL ₁ comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 31 The amino acid sequence represented by 32 has an amino acid sequence having at least 90% sequence identity.

In a specific embodiment of the present disclosure, the VH ₁ of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 33, and VL ₁ comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 33. The amino acid sequence shown in 34 has an amino acid sequence having at least 90% sequence identity.

According to some embodiments of the present disclosure, the second domain of the binding molecule of the present disclosure comprises a second heavy chain variable region VH ₂ and a second light chain variable region VL ₂ . The structure of the second domain is selected from Fab, Fab', F(ab') ₂ , Fv or scFv. For the first domain with Fv or scFv structure, the C-terminal of the VH ₂ peptide chain is directly connected to the N-terminal of the VL ₂ peptide chain or connected via a linker, and vice versa.

In some specific embodiments of the present disclosure, the VH ₂ includes: (e) HCDR1, HCDR2 and HCDR3 including the amino acid sequences shown in SEQ ID NO:13, 14 and 15 respectively; (f) including the amino acid sequences shown in SEQ ID NO:13, 14 and 15 respectively; HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in ID NOs: 19, 20 and 21; or, (g) HCDR1, HCDR2 and HCDR3 including the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively. In some specific embodiments of the present disclosure, the VL ₂ includes: (h) LCDR1, LCDR2 and LCDR3 including the amino acid sequences shown in SEQ ID NO: 16, 17 and 18; (i) including SEQ ID NO: 16, 17 and 18 respectively; LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in ID NOs: 22, 23 and 24; or, (j) LCDR1, LCDR2 and LCDR3 including the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively.

According to some specific embodiments of the present disclosure, the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 13, HCDR2 including the amino acid sequence shown in SEQ ID NO: 14, including the amino acid sequence shown in SEQ ID NO: 14 The HCDR3 of the amino acid sequence shown in ID NO:15, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:16, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:17, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:18 The amino acid sequence of LCDR3 is shown.

According to some specific embodiments of the present disclosure, the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 19, HCDR2 including the amino acid sequence shown in SEQ ID NO: 20, including the amino acid sequence shown in SEQ ID NO: 20 The HCDR3 of the amino acid sequence shown in ID NO:21, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:22, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:23, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:24 The amino acid sequence of LCDR3 is shown.

According to some specific embodiments of the present disclosure, the second structural domain includes: HCDR1 including the amino acid sequence shown in SEQ ID NO: 25, HCDR2 including the amino acid sequence shown in SEQ ID NO: 26, including the amino acid sequence shown in SEQ ID NO: 26 The HCDR3 of the amino acid sequence shown in ID NO:27, including the LCDR1 of the amino acid sequence shown in SEQ ID NO:28, including the LCDR2 of the amino acid sequence shown in SEQ ID NO:29, and including the LCDR2 of the amino acid sequence shown in SEQ ID NO:30 The amino acid sequence of LCDR3 is shown.

According to some embodiments of the present disclosure, the second domain of the binding molecule of the present disclosure comprises VH ₂ and VL ₂ . The VH ₂ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO:35, 37 or 39. The VL ₂ comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:36, 38 or 40.

In a specific embodiment of the present disclosure, the VH ₂ of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 35, and VL ₂ comprises an amino acid sequence identical to that shown in SEQ ID NO: 35. The amino acid sequence shown in 36 has an amino acid sequence having at least 90% sequence identity.

In a specific embodiment of the present disclosure, the VH ₂ of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 37, and VL ₂ comprises an amino acid sequence with the amino acid sequence shown in SEQ ID NO: 37. The amino acid sequence represented by 38 has an amino acid sequence having at least 90% sequence identity.

In particular embodiments of the present disclosure, the VH ₂ of the binding molecule of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 39, and VL ₂ comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 39. The amino acid sequence shown at 40 has an amino acid sequence having at least 90% sequence identity.

According to some embodiments of the present disclosure, in the binding molecules of the present disclosure, the first domain, the second domain and/or the third domain are directly connected or connected through a linker. According to some embodiments of the present disclosure, the first heavy chain variable region and the first light chain variable region of the first domain may be connected directly or through a linker. According to some embodiments of the present disclosure, the second heavy chain variable region and the second light chain variable region of the second domain are connected directly or through a linker.

In some embodiments of the present disclosure, the linker used in the binding molecule of the present disclosure has a sequence shown as (G _n S) _z , wherein n and z are each independently an integer of 1-4. For example, the linker sequence used in the present disclosure can be GS, GSGS (SEQ ID NO: 57), GGGGS (SEQ ID NO: 58), GGGGSGS (SEQ ID NO: 59), GGGGSGGGGSGGGGS (SEQ ID NO: 60) or Amino acid sequence shown by GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 61).

In some embodiments of the present disclosure, a binding molecule of the present disclosure comprises a first domain, a second domain and a third domain connected in the following order: first domain-third domain-second domain; Second domain-third domain-first domain; first domain-second domain-third domain; or, second domain-first domain-third domain.

According to some embodiments of the present disclosure, a binding molecule of the present disclosure may comprise two identical peptide chains that form a tetravalent homodimer. In some embodiments, one peptide chain of a binding molecule of the disclosure has an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41, 42, or 43.

In some embodiments, a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:44. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:45.

In some embodiments, the binding molecules of the present disclosure include heterotetramers that may include long peptide chains and short peptide chains, wherein the long peptide chains have at least 90 % sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:47.

In some embodiments, a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:48. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:49.

In some embodiments, a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:50. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:51.

In some embodiments, a binding molecule of the present disclosure may comprise a heterotetramer formed of a long peptide chain and a short peptide chain, wherein the long peptide chain has at least 90% of the amino acid sequence shown in SEQ ID NO:64. Sequence identity, the short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:51.

In some embodiments, a binding molecule of the present disclosure may comprise a heterohexamer formed of a long peptide chain, a first short peptide chain, and a second short peptide chain, wherein the long peptide chain has the same composition as that of SEQ ID NO:52. The amino acid sequence shown has at least 90% sequence identity, the first short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:54, and the second short peptide chain has at least 90% sequence identity with SEQ ID NO: The amino acid sequences shown at 53 have at least 90% sequence identity.

In some embodiments of the present disclosure, the binding molecules of the present disclosure may also include a heavy chain constant region, such as CH ₁ , CH ₂ , CH ₃ and/or CH ₄ . In some embodiments of the present disclosure, the binding molecules of the present disclosure may further comprise a light chain constant region CL.

Those skilled in the art can adjust the position and connection sequence of the above-mentioned functional fragments (eg, VH ₁ , VL ₁ , Fc, VH ₂ , VL _{1 ,} etc.) according to conventional techniques, while maintaining their respective binding properties.

In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding a binding molecule of the present disclosure or a fragment thereof.

In yet another aspect, the present disclosure provides a vector comprising the nucleic acid molecule described in the present disclosure. According to some embodiments of the present disclosure, the vector is an expression vector that can be transcribed and translated in a host cell to express the binding molecule of the present disclosure or a fragment thereof.

In yet another aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or vector of the present disclosure.

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the binding molecule of the present disclosure, the nucleic acid molecule of the present disclosure, the vector of the present disclosure or the host cell of the present disclosure and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure provides a method of treating or preventing cancer, comprising administering the pharmaceutical composition of the present disclosure to a subject in need thereof.

In yet another aspect, the present disclosure provides the use of a binding molecule of the present disclosure in the manufacture of a medicament for the treatment or prevention of cancer.

In some embodiments of the present disclosure, the cancer is selected from the group consisting of melanoma, glioma, kidney cancer, breast cancer, liver cancer, Wilms tumor, hepatoblastoma, blood cancer, and head and neck cancer.

In yet another aspect, the present disclosure provides the use of the binding molecule of the present disclosure, the nucleic acid molecule of the present disclosure, the vector of the present disclosure, the host cell of the present disclosure or the pharmaceutical composition of the present disclosure in the preparation of a medicament for treating or preventing cancer.

In this disclosure, the 4-1BB activation antibody is constructed in the form of Fab, scFv or single-chain antibody, and the linker sequence composed of flexible amino acids or the inherent linking amino acids of the antibody are fused to form a binding domain of the bispecific antibody, and then the GPC3-specific Sexual antibodies are constructed in the form of Fab, scFv or single-chain antibodies through the fusion of linkers composed of flexible amino acids or inherent linking amino acids of antibodies to form another binding domain of bispecific antibodies. The bispecific binding molecule for 4-1BB and GPC3 of the present disclosure can target and bind GPC3-positive tumor cells and at the same time generate an activation effect of T cells related to the 4-1BB signaling pathway. In addition, the bispecific binding molecules of the present disclosure exhibit excellent tumor growth inhibitory effects in mouse tumor models, and the combination of binding molecules targeting 4-1BB and GPC3 has a certain synergistic effect in inhibiting tumor growth.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present disclosure will become apparent from the following figures and detailed description of several embodiments, and also from the appended claims.

Description of drawings

In order to make the content of the present disclosure easier to understand, the present disclosure will be further described in detail below according to specific embodiments of the present disclosure and in conjunction with the accompanying drawings, wherein

Figure 1 shows the binding of binding molecules of the present disclosure to a CHO cell line stably transfected with human 4-1BB. Figure 1A The binding of the binding molecule of the present disclosure to the CHO cell line highly expressing human 4-1BB; Figure 1B The binding of the binding molecule of the present disclosure to the CHO cell line highly expressing human 4-1BB and the corresponding EC50 value. MFI indicates mean fluorescence intensity.

Figure 2 shows the binding of binding molecules of the present disclosure to recombinant human 4-1BB protein. Fig. 2A is the binding of the binding molecule of the present disclosure to the recombinant human 4-1BB protein; Fig. 2B is the binding of the binding molecule of the present disclosure to the recombinant human 4-1BB protein and the corresponding EC50 value.

Figure 3 shows the binding of binding molecules of the present disclosure to activated T cells.

Figure 4 shows the ELISA binding of binding molecules of the present disclosure to recombinant human GPC3 protein. FIG. 4A is the binding of the binding molecule of the present disclosure to the recombinant human GPC3 protein; FIG. 4B is the binding of the binding molecule of the present disclosure to the recombinant human GPC3 protein and the corresponding EC50 value.

Figure 5 shows the binding of binding molecules to human Huh7 and HepG2 liver cancer cell lines. Fig. 5A the binding of the binding molecule of the present disclosure to the human Huh7 liver cancer cell line; Fig. 5B the binding of the binding molecule of the present disclosure to the human HepG2 liver cancer cell line and the corresponding EC50 value.

Figure 6 shows that binding molecules of the present disclosure activate T cells in the presence of GPC3 positive liver cancer cells. (A) and (B) show that the binding molecule of the present disclosure stimulates T cells to secrete interleukins IL2 and IFNγ in the presence of GPC3-positive liver cancer cells Huh7, respectively. (C) to (F) respectively show that the binding molecule of the present disclosure stimulates T cells to secrete IL2 and IFNγ in the presence of GPC3-positive liver cancer cell HepG2. (G) shows that binding molecules of the present disclosure stimulate T cells to secrete IFNγ in the presence of GPC3-positive liver cancer cell Hep3B.

Figure 7 shows that binding molecules of the present disclosure inhibit the growth of GPC3 positive tumors. Figure 7A shows the results of inhibition of GPC3-positive tumor growth by 5 mg/kg of binding molecules; Figure 7B shows the inhibition of 20 mg/kg of binding molecules on the growth of GPC3-positive tumors.

Figure 8 shows a schematic representation of a binding molecule of the present disclosure.

Figure 9 shows that different concentrations of binding molecules of the present disclosure inhibit the growth of GPC3 positive tumors.

Figure 10 shows that different concentrations of HEC512-G1D inhibit the growth of GPC3 positive tumors.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in conjunction with embodiments. The specific embodiments described here are only used to explain the present disclosure, and are not intended to constitute any limitation to the present disclosure. Also, in the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concept of the present disclosure.

definition

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the meaning commonly understood by one of ordinary skill in the art. It should be noted that the terms used herein should be interpreted to have a meaning consistent with the context of this specification, and not be interpreted in an idealized or overly rigid manner.

The expression "about" as used herein is understood by those of ordinary skill in the art, and varies within a certain range depending on the context in which it is used. If the use of this term is not understood by one of ordinary skill in the art depending on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

As used herein, "4-1BB", also known as CD137, TNFRSF9, etc., is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRS). 4-1BB is a co-stimulatory receptor expressed on various cells of the immune system, especially on CD8+ T cells. Due to its ubiquitous expression, and the ability of 4-1BB to enhance potent and long-lasting immune effects, 4-1BB has become a clinical target for cancer immunotherapy.

As used herein, "glypican 3" or "GPC3" is used interchangeably and is a carcinoembryonic antigen belonging to the glypican family, which is composed of glycosyl Heparan sulfate proteoglycans anchored by phosphatidylinositol. Glypican regulates the activity of several growth factors including Wnts, Hedgehogs, bone morphogenetic proteins and fibroblast growth factor (FGF). Glypican is characterized by covalent linkages to polysaccharide chains known as heparan sulfate glycosaminoglycans. Glypican participates in cell signaling at the cell-extracellular matrix interface. To date, six distinct members of the human glypican family have been identified. Cell membrane-bound GPC3 consists of two subunits linked by one or more disulfide bonds. GPC3 is expressed in the developing fetal liver and placenta and is downregulated or silenced in normal adult tissues. GPC3 is highly expressed in various cancers, especially hepatocellular carcinoma (HCC), melanoma, Wilms tumor and hepatoblastoma.

As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, carboxyl, amino and R groups, such as homoserine, norleucine, methionine sulfoxide, Methylsulfonium methionine. These analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are compounds that differ structurally from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. As used herein, "polar amino acid" refers to an amino acid comprising a side chain that prefers to reside in an aqueous environment. In some embodiments, the polar amino acid is selected from arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and tyrosine. Polar amino acids can be positively charged, negatively charged, or neutrally charged. As used herein, "non-polar amino acid" is selected from the group consisting of alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan acid and valine.

"Substitution with one or more different amino acids" as used herein means that at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) is replaced by another different "replacement" amino acid residue . "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While inserts typically consist of insertions of 1 or 2 amino acid residues, larger "peptide insertions" can also be made, for example insertions of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues may be naturally occurring or non-naturally occurring. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

Antibodies suitable for use in the present disclosure may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as alanine, valine , leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, an essential or nonessential amino acid residue in an antibody is preferably replaced with another amino acid residue from the same side chain family. In certain embodiments, amino acid stretches may be replaced with structurally similar stretches that differ in the order and/or composition of side chain family members. Alternatively, in certain embodiments, mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into the binding polypeptides of the invention and directed against these binding polypeptides The ability to bind to the desired target is screened.

As used herein, the term "anti-4-1BB agonistic antibody" or "specific agonistic antibody against 4-1BB" means that it specifically binds to 4-1BB and partially or completely promotes, induces, increases and/or activates Antibodies to 4-1BB biological activity, responses and/or downstream pathways mediated by 4-1BB signaling or other 4-1BB mediated functions. Tumor necrosis factor receptor (TNFR) signaling, particularly TNFRSF activation, requires receptor clustering and multimerization. 4-1BB is one of the TNFSF receptors known to require clustering to trigger downstream signaling. Formation of 2 or more trimers by cross-linking of 4-1BB has been reported to result in a stronger activated protein. In some embodiments, the anti-4-1BB agonistic antibody binds 4-1BB and induces multimerization of 4-1BB into 2 or more trimers.

The terms "full-length antibody" and "intact antibody" of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody. "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) ( Also known as the heavy chain constant region). From N-terminus to C-terminus, each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region). The heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), which can be further divided into Subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1 ) and α2 (IgA2). The light chains of an antibody, based on the amino acid sequence of their constant domains, can be of one of two types, kappa and lambda light chains.

Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The binding molecules of the present disclosure are homodimers comprising two identical peptide chains bonded by disulfide bonds. In some embodiments, the two identical peptide chains are long peptide chains comprising a first domain and a second domain that specifically bind 4-1BB and GPC3, respectively. The long peptide chain of the binding molecule of the present disclosure may include VL ₂ -VH ₂ -Fc-VH ₁ -VL ₁ , VH ₂ -VL ₂ -Fc-VH ₁ -VL ₁ , VL ₂ -VH ₂ - Fc- _VL1 - _VH1 , VH1 _- VL1-Fc-VH2- _VL2 _, _VL1 - _VH1 -Fc- _VL2 _- _VH2 , VL1 _- _VH1 -Fc- _VH2 - _VL2 , or Other connection methods known to those skilled in the art, wherein each fragment may or may not have the linker sequence disclosed herein.

The binding molecule of the present disclosure is a heterotetramer, which includes two long peptide chains bonded by disulfide bonds, and two short peptide chains respectively bonded to the corresponding long peptide chains by disulfide bonds. In some embodiments, the long peptide chain may include VH ₂ -Fc-VH ₁ -VL ₁ from the N-terminus, and the short peptide chain may include VL ₂ from the N-terminus. In some embodiments, the long peptide chain may include VH ₁ -Fc-VH ₂ -VL ₂ from the N-terminus, and the short peptide chain may include VL ₁ from the N-terminus. In some embodiments, the long peptide chain may include VH ₁ -Fc-VL ₂ -VH ₂ from the N-terminus, and the short peptide chain may include VL ₁ from the N-terminus. Alternatively, the functional fragments in the long peptide chain and the short peptide chain can be combined in other ways known to those skilled in the art.

The binding molecule of the present disclosure is a heterohexamer comprising two long peptide chains, two first short peptide chains and two second short peptide chains, wherein the two long peptide chains are bonded by disulfide bonds, The first short peptide chain and the second short peptide chain are respectively bonded to corresponding regions of the long peptide chain through disulfide bonds. In some embodiments, the long peptide chain may include VH ₁ -Fc-VL ₂ , the first short peptide chain includes VL ₁ , and the second short peptide chain includes VH ₂ . Alternatively, the functional fragments in the long peptide chain and the short peptide chain can be combined in other ways known to those skilled in the art.

The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" as used herein refer to the binding of an antibody or antigen-binding portion thereof to an epitope on a predetermined antigen. Typically, when assayed by surface plasmon resonance (SPR) technique in a BIACORE 2000 instrument using recombinant human 4-1BB as the analyte and the antibody as the ligand, the antibody is present at about less than 10 ⁻⁶ M, such as about less than 10 ^-7 M, 10 ⁻⁸ M, 10 ⁻⁹ M or 10 ⁻¹⁰ M or even lower equilibrium dissociation constant (K _D ) binding, and binding to the predetermined antigen affinity is binding to other than the predetermined antigen or closely related At least twice the affinity of non-specific antigens (eg, BSA, casein) outside the antigen.

The term "subject" as used herein includes any human or non-human animal. For example, the methods and compositions of the invention can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

As used herein, the terms "antibody fragment", "antigen-binding fragment", "antigen-binding portion" or similar terms refer to an antibody that retains binding to a target antigen (e.g., 4-1BB or GPC3) and has the same activity as the full-length antibody. fragment. Such fragments include, for example, single chain antibodies, single chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments or F(ab')2 fragments. A scFv fragment is a single polypeptide chain that includes the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, tribodies and diabodies are included within the definition of antibody and are suitable for use in the methods described herein. See eg Todorovska et al. (2001), J. Immunol. Methods, 248(1): 47-66; Hudson and Kortt, (1999), J. Immunol. Methods, 231(1): 177-189; Poljak, ( 1994), Structure, 2(12):1121-1123; Rondon and Marasco, (1997), Annu.Rev.Microbiol., 51:257-283, the respective disclosures of which are incorporated herein by reference in their entirety middle.

The term "Fc fragment" as used herein refers to the constant region of a full-length immunoglobulin. In some embodiments, "Fc fragment" refers to the last two constant domains (CH2-CH3) of IgA, IgD, IgG, or the last three constant domains (CH2-CH3-CH4) of IgE and IgM, and N-terminal flexible hinges of these domains.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), where consideration is given to optimally aligning the two sequences The number of slots to introduce for alignment and the length of each slot. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and

length weight

1, 2, 3, 4, 5 or 6 determination. The percent identity between two nucleotide or amino acid sequences can also be calculated using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), using the PAM120 weight residue table, gap length penalty 12 and a gap penalty of 4, the algorithm has been incorporated into the ALIGN program (version 2.0). Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970) ), using the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and

length weights

1, 2, 3, 4, 5, or 6 determined, the algorithm has been incorporated into the GCG software package (available from http://www.gcg.com ) in the GAP program.

The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" as used herein means that certain segments of the variable domains generally differ in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.

As used herein, the term "hypervariable region" or "HVR" refers to the region of an antibody variable domain region that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)", which have the highest sequence variability and/or are involved in antigen recognition. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26-L32(L1), L50-L52(L2), L91-L96(L3), H26-H32( H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L24-L34(L1), L50-L56(L2), L89-L97(L3), H31-H35( H1), H50-H65 (H2) and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). based on

Defining the rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32(L1), L50-L51(L2), L89-L97(L3), H26-H33(H1) , H51-H56(H2) and H93-H102(H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). For comparison, in Table 1 are listed the corresponding amino acid residues comprising the CDRs defined in the references cited above. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in various ways, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the position of the structural loop region, and antibody humanization based on CDR grafting A reference tool for design (see J Mol Biol 273:927-48, 1997). It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing the above-mentioned existing ones as described by the present invention. Complementarity-determining regions defined in any one of the known schemes. Although the scope of protection claimed in the present disclosure is based on the sequence shown by the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

Table 1. Amino acid numbering system for each CDR region (see http://bioinf.org.uk/abs/)

CDR\编号体系CDR\numbering system	KabatKabat	ChothiaChothia	IMGTIMGT
LCDR1LCDR1	24-3424-34	26-3226-32	27-3227-32
LCDR2LCDR2	50-5650-56	50-5250-52	50-5150-51
LCDR3LCDR3	89-9789-97	91-9691-96	89-9789-97
HCDR1HCDR1	31-3531-35	26-3226-32	26-3326-33
HCDR2HCDR2	50-6550-65	52-5652-56	51-5651-56
HDCR3HDCR3	95-10295-102	96-10196-101	93-10293-102

"Framework" or "FR" as used herein refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

According to some embodiments of the present disclosure, a binding molecule of the present disclosure may comprise a Fab arm comprising one heavy chain-light chain pair that specifically binds an antigen. According to some embodiments of the present disclosure, the binding molecules of the present disclosure may include recombinant IgG-like dual targeting molecules, wherein each of the two sides of the molecule contains Fab fragments or parts of Fab fragments of at least two different antibodies; IgG fusion molecules, wherein A full-length IgG antibody fused to an additional Fab fragment or a portion of a Fab fragment; an Fc fusion molecule in which a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region, or a portion thereof; a Fab fusion molecule in which Different Fab fragments are fused together; scFv and diabodies based heavy chain antibodies (e.g. domain antibodies, nanobodies) where different single chain Fv molecules or different diabodies or different heavy chain antibodies are fused to each other or Fusion with another protein or carrier molecule.

The "class" of an antibody as used herein refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The "humanized antibody" used herein comprises the amino acid residues from the non-human hypervariable region (HVR) and the constant regions of the heavy chain and light chain from the human antibody spliced to obtain the complete sequence of the humanized antibody. In certain embodiments, a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

As used herein, "cross-reactivity" refers to the ability of an antibody of the present disclosure to bind 4-1BB or GPC3 from a different species. For example, an antibody of the disclosure may bind human 4-1BB while also binding 4-1BB of another species (eg, mouse, rat, cynomolgus monkey, or dog). As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (eg, SPR, ELISA), or with physiologically expressed cells that bind or otherwise functionally interact.

As used herein, having "at least 90% sequence identity" with a sequence compared to a sequence may include at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% %, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

The "expression vector" and "expression construct" used herein can be used interchangeably. When the above-mentioned isolated nucleic acid molecule is connected to the carrier, the nucleic acid sequence can be directly or indirectly connected to the regulatory elements on the carrier, as long as these regulatory elements It is sufficient that the element can regulate the translation and expression of the nucleic acid molecule. Of course, these regulatory elements may come directly from the vector itself, or be exogenous, that is, not from the vector itself. That is, a nucleic acid molecule is operably linked to a regulatory element. In this paper, "operably linked" refers to linking the exogenous gene to the carrier, so that the regulatory elements in the carrier, such as transcriptional regulatory sequences and translational regulatory sequences, etc., can play their intended role in regulating the transcription and translation of the exogenous gene function. Of course, the polynucleotides used to encode the heavy chain and light chain of the antibody can be independently inserted into different vectors, usually inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages and the like.

The "cell" or "recombinant cell" used herein may contain an expression vector. Expression vectors can be introduced into mammalian cells to obtain recombinant cells, which are then used to express the antibodies or antigen-binding portions provided in the present disclosure. The corresponding antibody can be obtained by culturing the recombinant cells. These usable mammalian cells are, for example, CHO cells and the like.

As used herein, "pharmaceutically acceptable carrier" may include any solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, the pharmaceutical composition may contain isotonic agents, such as sugars, polyalcohols (such as mannitol, sorbitol) or sodium chloride, etc. Of course, pharmaceutically acceptable carriers may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, to prolong the shelf life or potency of the antibody.

For example, an antibody of the disclosure, or an antigen-binding portion thereof, can be incorporated into a pharmaceutical composition suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (such as injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, Powders, liposomes and suppositories. Typical pharmaceutical compositions are in the form of solutions for injection or infusion. Antibodies of the disclosure, or antigen-binding portions thereof, can be administered by intravenous infusion, injection, intramuscular or subcutaneous injection.

A "therapeutically effective amount" of a drug (eg, a pharmaceutical composition) refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect. For example, a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.

The term "blood cancer" as used herein includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B-cell lymphoma (B-cell blood cancer) and T-cell lymphoma. B-cell lymphomas include Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphoma include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma (MCL), Burkitt Lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, lymph node marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granuloma. Non-limiting examples of T cell lymphoma include extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma. Hematological malignancies also include leukemias such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematologic malignancies also include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematological malignancies encompasses other blood and/or B or T cell related cancers.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. In the following description, descriptions of well-known technologies are omitted to avoid unnecessarily obscuring the concept of the present disclosure. Such techniques are described in a number of publications, such as "A Laboratory Guide to Molecular Cloning, Fourth Edition" (Cold Spring Harbor Laboratory Scientific Press).

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1. Transient transfection of HEK293 suspension cells to obtain binding molecules.

Using the whole gene synthesis method, the peptide chains in Table 2 below were subjected to whole gene synthesis and inserted into the pcDNA3.1 vector respectively. The constructed vectors were transiently transfected into HEK293 cells for protein expression. In addition, for binding molecules containing multiple peptide chains, the vectors inserted with multiple peptide chains are co-transfected into HEK293 cells (for example, peptide chain 1, peptide chain 2 and peptide chain 2 of IOB1-FabIg-S2 are respectively inserted Peptide 3 vector, co-transfected into HEK293 cells).

Table 2. The structure and sequence corresponding to each polypeptide sequence

Note: the heavy chain variable region and the light chain variable region of anti-4-1BB are respectively derived from monoclonal antibody HEC511 (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 67, and the amino acid sequence of the light chain is shown in SEQ ID NO: 54) or monoclonal antibody HEC512 (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 68, and the amino acid sequence of the light chain is shown in SEQ ID NO: 66). The heavy chain variable region and the light chain variable region of anti-GPC3 are derived from three monoclonal antibodies HS20 respectively (the sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37) ID NO: 38), 4A6 (the sequence of the heavy chain variable region is shown in SEQ ID NO35, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36) or GC33 (the sequence of the heavy chain variable region is shown in SEQ ID NO: 36) The sequence is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:40).

The expressed binding molecules were purified by ProteinA medium, and the binding molecules IOB1-S, IOB1-M, IOB1-ScFvIg-S, IOB1-FabIg-S2, IOB1-G, IOB1-G-Fab, IOB1-G, IOB1-G-Fab, The structure diagram of IOB1-G-Fab1, IOB1-G-Fab2 and IOB1-G1D-Fab2 is shown in FIG. 8 . These binding molecules obtained were used for the following assay evaluation.

The expression titers of the above-mentioned binding molecules obtained in each 100 mL suspension cell culture medium were measured, and the results are shown in Table 2. In Table 2, Isotype (isotype) IgG served as a negative control.

Table 2. Titers of each binding molecule per 100 mL of suspension cell culture medium.

Example 2. Binding of the binding molecule of the present disclosure to a CHO cell line stably transfected with human 4-1BB.

In this example, flow cytometry was used to detect the binding ability of the binding molecule to a CHO cell line stably transfected with human 4-1BB. After the binding molecules were incubated with the cells, FITC-labeled goat anti-mouse IgG Fc secondary antibody (Abcam: ab97023) was used to bind, GC33 and isotype IgG antibodies were used as negative controls, and the corresponding antibody HEC511 (heavy chain The amino acid sequence of the antibody is shown in SEQ ID NO:67, the amino acid sequence of the light chain is shown in SEQ ID NO:54) and antibody HEC512 (the amino acid sequence of the heavy chain is shown in SEQ ID NO:68, and the amino acid sequence of the light chain is shown in Shown in SEQ ID NO:66) as positive control sample. The results are shown in Figure 1. It can be seen from Figure 1 that the binding molecule can effectively bind to the protein expressed on the human 4-1BB (UniProtKB-Q07011) membrane.

Example 3. Binding of binding molecules of the present disclosure to human 4-1BB recombinant protein

In this example, ELISA was used to detect the binding ability of the binding molecule to human 4-1BB (UniProtKB-Q07011) recombinant protein. Coat the recombinant 4-1BB protein on the ELISA plate overnight at 4°C, wash it three times and then dry it, block it at 37°C for 1 hour, wash it three times and dry it for later use. Incubate the binding molecule of the present disclosure with the above-mentioned spare ELISA plate at 4°C for 2 hours, wash it three times and then dry it, then use HRP-labeled goat anti-mouse IgG-Fc secondary antibody to bind, and use the isotype IgG antibody as a negative control , and the corresponding antibodies HEC511 and HEC512 used to construct the binding molecules were used as control samples. The results are shown in Figure 2.

It can be seen from FIG. 2 that the binding molecule of the present disclosure can effectively bind to the human 4-1BB (UniProtKB-Q07011) recombinant protein.

Example 4. Binding of binding molecules of the present disclosure to T cells isolated and activated from human blood in vitro

T cells were isolated from human-derived PBMC cells, and CD3 and CD28 antibody-coupled magnetic beads were used to stimulate T cells in vitro for 48-72 hours. The stimulated T cells could highly express 4-1BB protein. Then, the ability of the binding molecule of the present disclosure to bind to the stimulated T cells was detected by flow cytometry. After incubation of the binding molecules with the cells, subsequent binding was performed using a FITC-labeled goat anti-mouse IgG Fc secondary antibody, and an isotype IgG antibody was used as a negative control. The results are shown in FIG. 3 .

It can be seen from Figure 3 that the binding molecules of the present disclosure can effectively bind to activated human T cells. That is to say, the binding molecules of the present disclosure can effectively bind to the 4-1BB protein expressed by activated T cells.

Example 5. Binding of binding molecules of the present disclosure to human GPC3 recombinant protein

In this example, ELISA was used to detect the binding ability of the binding molecule of the present disclosure to human GPC3 (XP_022382036.1) recombinant protein. The recombinant GPC3 protein was coated on the ELISA plate overnight at 4°C, washed three times and then dried, blocked at 37°C for 1 hour, washed three times and then dried for later use. Incubate the antibody with the above spare ELISA plate at 4°C for 2 hours, wash it three times and then dry it, then use HRP-labeled goat anti-mouse IgG Fc secondary antibody to bind, use antibody HEC511, isotype IgG antibody as negative control, and use The antibodies HS20 and GC33 corresponding to the fusion protein were used as control samples. The results are shown in FIG. 4 .

It can be seen from Fig. 4 that the binding molecule of the present disclosure can effectively bind to the human GPC3 (XP_022382036.1) recombinant protein.

Example 6. Binding of binding molecules of the present disclosure to human liver cancer cell lines

It is known that GPC3 protein is highly expressed in liver cancer cells. In order to detect whether the binding molecules of the present disclosure can bind to liver cancer cells, conventionally cultured Huh7 or HepG2 liver cancer cells were digested with trypsin, collected, and flow cytometry was used to detect whether the binding molecules of the present disclosure could bind to human liver cancer cell lines Huh7 or HepG2. Binding capacity of HepG2. After incubating the binding molecule of the present disclosure with the cells, FITC-labeled goat anti-mouse IgG Fc secondary antibody was used to bind, the isotype IgG antibody was used as a negative control, and the corresponding antibodies GC33 and HEC511 used to construct the fusion protein were used as control samples. The results are shown in FIG. 5 .

It can be seen from FIG. 5 that the binding molecule of the present disclosure can effectively bind to the GPC3 protein expressed by the human liver cancer cell line.

Example 7. Affinity of binding molecules of the present disclosure to human 4-1BB recombinant protein.

In this example, the binding affinity of the binding molecules of the present disclosure to 4-1BB was determined by using the biofilm light interference technique (Bio-Layer Interferometry, BLI). To put it simply, the human 4-1BB (UniProtKB-Q07011) protein was immobilized on the sensor for 300s, then the sensor was put into the PBST solution to balance the baseline for 180s, and then the probe was immersed in the fusion protein solution for 600s , and then the probe was placed in PBST solution to dissociate for 1200s, and finally the probe was placed in the regeneration solution to complete the regeneration and preservation of the probe. The binding affinity of the binding molecule of the present disclosure to the human recombinant protein 4-1BB was detected by BLI detection. The results are shown in Table 3.

Table 3. Binding affinity of binding molecules of the present disclosure to human recombinant 4-1BB protein

Example 8. Binding Affinity of the Binding Molecules of the Disclosure to the Human GPC3 Recombinant Protein

In this example, the binding affinity of the binding molecule of the present disclosure to GPC3 was measured by using bio-layer interferometry (Bio-Layer Interferometry, BLI). To put it simply, the human GPC3 (XP_022382036.1) protein was immobilized on the sensor for 300s, then the sensor was put into the PBST solution to balance the Baseline180s, then the probe was immersed in the fusion protein solution for 600s, and then The probe was placed in PBST solution for 1200s, and finally the probe was placed in the regeneration solution to complete the regeneration and preservation of the probe. Using BLI detection, the binding affinity of the binding molecule of the present disclosure to the human recombinant protein GPC3 was determined. The results are shown in Table 4.

Table 4. Binding affinity of binding molecules of the present disclosure to human recombinant GPC3 protein

Example 9. Binding molecules of the present disclosure activate T cells in a GPC3 positive liver cancer cell dependent manner.

Coat the CD3 antibody on the microtiter plate to evenly cover a certain density of tumor cells. After human T cells are isolated, the T cells and the binding molecule of the present disclosure are added to a microtiter plate, and the expression level of IL2 in the supernatant is detected after three days, or the expression level of IFN-γ in the supernatant is detected after five days. HEC511, HS20, 4A6 or isotype IgG antibodies were used as controls. The result is shown in Figure 6.

Figures 6A and 6B show that binding molecules of the present disclosure can stimulate T cells to secrete interleukins IL2 and IFNγ in the presence of GPC3-positive liver cancer cells Huh7. 6C-6F show that the binding molecules of the present disclosure can stimulate T cells to secrete IL2 and detect IFNγ in the presence of GPC3-positive liver cancer cells HepG2. FIG. 6G shows that the binding molecules of the present disclosure can stimulate T cells to secrete IFNγ in the presence of GPC3-positive liver cancer cells Hep3B.

Example 10. Verification of the tumor suppressive effect of the binding molecules of the present disclosure in the tumor-bearing humanized mouse model of MC38 high-expression GPC3 stably transfected cell line

The MC38 tumor cell line (MC38-hGPC3 cells) resuspended in PBS with high expression of human GPC3 protein was inoculated in B- ^h4-1BB /h4-1BBL human-derived Subcutaneously on the right side of the mouse. When the average tumor volume reached 100-150 mm ³ , the appropriate mice were selected according to the tumor volume and body weight of the mice, and were evenly distributed into 3 experimental groups, with 6 mice in each group. The samples of each group were intraperitoneally administered at a dose of 5 mg/kg (the control group was given PBS), twice a week. After grouping, the tumor volume was measured twice a week with a vernier caliper. Before euthanasia, the tumor volume was measured, and the long and short diameters of the tumor were measured. The volume calculation formula was: tumor volume=0.5×long diameter×short diameter ² . The results are shown in Figure 7A. It can be seen that the bispecific antibody of the present disclosure can significantly inhibit the growth of tumor after administration; and at the same dose, the onset time of bispecific antibody inhibition of tumor is earlier than that of anti-4-1BB monoclonal Anti-41BB monoclonal HEC512 also significantly earlier eliminated tumors.

Example 11 Evaluation of anti-tumor activity of the tested antibody in the tumor-bearing humanized mouse model of MC38 high-expression GPC3 stably transfected cell line

The right side of 41BB/41BBL double humanized C57BL/66 mice was subcutaneously inoculated with 5×10 ⁵ cells/0.1 mL/mouse, and the tumor cell line MC38 highly expressing human GPC3 protein was inoculated. When the average tumor volume reaches 100-150mm ³ , select suitable mice according to their body weight and tumor volume, inject the test antibody IOB1-G-Fab2 into the mice at a dose of 20 mg/kg intraperitoneally, and measure the tumor regularly Size, tumor growth or inhibition was observed. The results are shown in FIG. 7B , showing that the tested antibody has the effect of significantly inhibiting the growth of GPC3-positive tumors.

Example 12. Anti-tumor activity evaluation of the tested antibody in the tumor-bearing humanized mouse model of MC38 high expression GPC3 stably transfected cell line

The right side of 41BB/41BBL double humanized C57BL/66 mice was subcutaneously inoculated with 5×10 ⁵ cells/0.1 mL/mouse, and the tumor cell line MC38 highly expressing human GPC3 protein was inoculated. When the average tumor volume reaches ^100-150mm , select suitable mice according to mouse body weight and tumor volume to enter the group, test antibody IOB1-G1D-Fab2 and HEC512-G1D (the amino acid sequence of the heavy chain is as SEQ ID NO: 65, the amino acid sequence of the light chain is shown in SEQ ID NO: 66) according to 20mg/kg, 5mg/kg and 1mg/kg doses of intraperitoneal injection into mice, regularly measure the size of the tumor, observe the growth or inhibition of the tumor . The results are shown in Figures 9 and 10, showing that the tested antibodies significantly inhibited the growth of GPC3-positive tumors, and the inhibitory effect of IOB1-G1D-Fab2 was better than that of HEC512-G1D.

The technical solution of the present disclosure is not limited to the limitations of the above specific embodiments, and all technical modifications made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

A binding molecule, characterized in that the binding molecule comprises:

a first domain, wherein the first domain specifically binds 4-1BB or a fragment thereof, and

A second domain that specifically binds Glypican 3 (GPC3) or a fragment thereof.
The binding molecule according to claim 1, wherein the binding molecule further comprises a third domain, and the third domain comprises an Fc fragment of an immunoglobulin,

Preferably, the immunoglobulin is selected from IgA, IgG, IgM, IgD and IgE, preferably IgG, more preferably IgG1, IgG2, IgG3 or IgG4;

Preferably, the Fc fragment has one or more mutations among L234F, L235E, P331S, D356E and L358M, wherein the Fc fragment is numbered according to the EU index of Kabat;

More preferably, the third domain comprises the amino acid sequence shown in SEQ ID NO: 55, 62 or 69 or a variant thereof.
The binding molecule according to claim 1 or 2, wherein the first domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1 , wherein:

The VH 1 includes:

(a) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 1, 2 and 3, respectively; or

(b) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO:7, 8 and 9, respectively,

The VL 1 includes:

(c) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 4, 5 and 6, respectively; or

(d) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 10, 11 and 12, respectively.
The binding molecule according to any one of claims 1 to 3, characterized in that,

The VH1 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 or 33, and/or

The VL 1 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:32 or 34.
The binding molecule according to any one of claims 1 to 4, wherein the second domain comprises a second heavy chain variable region VH 2 and a second light chain variable region VL 2 , wherein

The VH 2 includes:

(e) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14 and 15, respectively;

(f) HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively; or

(g) HCDR1, HCDR2 and HCDR3 comprising the amino acid sequences shown in SEQ ID NO: 25, 26 and 27, respectively,

The VL 2 includes:

(h) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 16, 17 and 18, respectively;

(i) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 22, 23 and 24, respectively; or

(j) LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences shown in SEQ ID NO: 28, 29 and 30, respectively.
The binding molecule according to any one of claims 1 to 5, characterized in that,

The VH 2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 35, 37 or 39, and/or

The VL 2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:36, 38 or 40.
The binding molecule according to any one of claims 1 to 6, wherein the structure of the first domain and/or the second domain is selected from the group consisting of Fab, Fab', F(ab') 2 , Fv or scFv.
The binding molecule according to any one of claims 1 to 7, wherein the first domain, the second domain and/or the third domain are connected directly or via a linker,

The first heavy chain variable region and the first light chain variable region of the first domain are connected directly or via a linker, and/or

The second heavy chain variable region and the second light chain variable region of the second domain are connected directly or through a linker.
The binding molecule according to any one of claims 1 to 8, wherein the linker has a sequence shown as (G n S) z , wherein n and z are each independently an integer of 1-4.
The binding molecule according to any one of claims 1 to 9, wherein the binding molecule comprises a first domain, a second domain and a third domain connected in the following order:

First domain-third domain-second domain;

Second domain-third domain-first domain;

First domain-second domain-third domain; or,

Second domain - first domain - third domain.
The binding molecule according to any one of claims 1 to 10, wherein the binding molecule comprises a homodimer formed by a peptide chain,

Preferably, the peptide chain has an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 41, 42 or 43.
The binding molecule according to any one of claims 1 to 10, wherein the binding molecule comprises a heterotetramer formed by a long peptide chain and a short peptide chain, wherein

(i) the long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:44, and

The short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:45;

(ii) the long peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:46, and

The short peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:47;

(iii) the long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:48, and

The short peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 49; or

(iv) the long peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:50, and

The short peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:51, or

(v) the long peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:64, and

The short peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:51.
The binding molecule according to any one of claims 1 to 10, wherein the binding molecule comprises a heterohexamer formed by a long peptide chain, a first short peptide chain and a second short peptide chain, wherein

The long peptide chain has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:52,

The first short peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:54, and

The second short peptide chain has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:53.
An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes the binding molecule according to any one of claims 1-13.
A vector, characterized in that the vector comprises the nucleic acid molecule according to claim 14, and the vector is preferably an expression vector.
A host cell, characterized in that the host cell comprises the nucleic acid molecule of claim 14 or the vector of claim 15.
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the binding molecule as claimed in any one of claims 1 to 13, the nucleic acid molecule as claimed in claim 14, the carrier as claimed in claim 15 or The host cell and pharmaceutically acceptable carrier as claimed in claim 16.
A binding molecule as claimed in any one of claims 1 to 13, a nucleic acid molecule as claimed in claim 14, a carrier as claimed in claim 15, a host cell as claimed in claim 16 or as claimed in claim 17 The application of the pharmaceutical composition in the preparation of a drug for treating or preventing cancer, preferably, the cancer is selected from melanoma, glioma, renal cancer, breast cancer, liver cancer, Wilms tumor, hepatoblastoma, Blood cancers and head and neck cancers.